Name: retrograde neuroanatomical tracing of phrenic motor neurons in mice
Uploaded: 2018-02-22T14:00:00
Description: Watch this Scientific Journal Video about Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice at JoVE.com

We are grateful to Robert Graffin and Pauline Duhant for their technical support.

The experimental protocol was conducted in compliance with the European Communities Council Directives for Animal Experiment (2010/63/EU, 86/609/EEC and 87-848/EEC) and was approved by the Animal Ethics Committee of University of Namur (ethic project n&#176;17-284). Figure 1 depicts the two respective approaches: transdiaphragmatic or transthoracic injections. Use male C57bl/6J mice (n=18), aged from 3 to 4 months in the study.
1. Preparation of CTB Solution
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<ol>
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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rafting+with+cholera+toxin:+endocytosis+and+trafficking+from+plasma+membrane+to+ER.">Rafting with cholera toxin: endocytosis and trafficking from plasma membrane to ER.</a> FEMS Microbiol Lett. 266 (2), 129-137 (2007).</li><li>Fujinaga, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gangliosides+that+associate+with+lipid+rafts+mediate+transport+of+cholera+and+related+toxins+from+the+plasma+membrane+to+endoplasmic+reticulm.">Gangliosides that associate with lipid rafts mediate transport of cholera and related toxins from the plasma membrane to endoplasmic reticulm.</a> Mol Biol Cell. 14 (12), 4783-4793 (2003).</li><li>Badizadegan, K., Wheeler, H. E., Fujinaga, Y., Lencer, W. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trafficking+of+cholera+toxin-ganglioside+GM1+complex+into+Golgi+and+induction+of+toxicity+depend+on+actin+cytoskeleton.">Trafficking of cholera toxin-ganglioside GM1 complex into Golgi and induction of toxicity depend on actin cytoskeleton.</a> Am J Physiol Cell Physiol. 287 (5), C1453-C1462 (2004).</li><li>Abbott, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+axonal+transport+in+the+rat+visual+pathway.">Imaging axonal transport in the rat visual pathway.</a> Biomed Opt Express. 4 (2), 364-386 (2013).</li><li>Li, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+the+astrocyte+glutamate+transporter+GLT1+exacerbates+phrenic+motor+neuron+degeneration,+diaphragm+compromise,+and+forelimb+motor+dysfunction+following+cervical+contusion+spinal+cord+injury.">Overexpression of the astrocyte glutamate transporter GLT1 exacerbates phrenic motor neuron degeneration, diaphragm compromise, and forelimb motor dysfunction following cervical contusion spinal cord injury.</a> J Neurosci. 34 (22), 7622-7638 (2014).</li><li>Lepore, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraspinal+cell+transplantation+for+targeting+cervical+ventral+horn+in+amyotrophic+lateral+sclerosis+and+traumatic+spinal+cord+injury.">Intraspinal cell transplantation for targeting cervical ventral horn in amyotrophic lateral sclerosis and traumatic spinal cord injury.</a> J Vis Exp. (55), (2011).</li><li>Llado, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degeneration+of+respiratory+motor+neurons+in+the+SOD1+G93A+transgenic+rat+model+of+ALS.">Degeneration of respiratory motor neurons in the SOD1 G93A transgenic rat model of ALS.</a> Neurobiol Dis. 21 (1), 110-118 (2006).</li><li>Lepore, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+transplantation-based+astrocyte+replacement+is+neuroprotective+in+a+model+of+motor+neuron+disease.">Focal transplantation-based astrocyte replacement is neuroprotective in a model of motor neuron disease.</a> Nat Neurosci. 11 (11), 1294-1301 (2008).</li><li>Sieck, G. C., Fournier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diaphragm+motor+unit+recruitment+during+ventilatory+and+nonventilatory+behaviors.">Diaphragm motor unit recruitment during ventilatory and nonventilatory behaviors.</a> J Appl Physiol. 66 (6), 2539-2545 (1989).</li><li>Janicot, M., Clot, J. 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The protocol described herein can be applied to any strain of adult mice or to any experimental paradigm in which the integrity of the diaphragm-PhMN circuitry should be evaluated. For instance, amyotrophic lateral sclerosis (ALS) and cervical spinal cord injury (cSCI) are conditions associated with PhMN loss, anterograde degeneration of phrenic axons and subsequent respiratory compromise. Animal models of ALS or cSCI mimic histopathological and functional respiratory deficits observed in human diseases. In these models,...

The aim of the study is to present a reliable method to identify phrenic motor neurons (PhMN) on mouse spinal cord sections. Injection of a fluorescent neuroanatomical tracer in the pleural cavity was chosen as the delivery method to reach the phrenic neuromuscular projections onto the diaphragm and use retrograde transport along the phrenic axons to label phrenic cell bodies. Two techniques of intrapleural delivery are described: transdiaphragmatic versus transthoracic.
Phrenic motor neurons are spinal relay cells whose axons converge into phrenic nerves, which ultimately innervate the diaphragm. These are lower motor neurons receiving the inspiratory drive from the bulbar respiratory centers and relaying it to the diaphragm neuro-muscular junctions (NMJ). PhMN are structured into two motor columns, one for each hemicord, running along the mid-cervical spine. In most of mammalian species including humans, the phrenic motor columns spread from levels C3 to C61,2,3. We and others have confirmed that PhMN concentrated in C3-C5 levels in rat and mouse spinal cord4,5,6,7,8. The topographical distribution of phrenic cells is not at random; motor neurons innervating the sternal part of the diaphragm are distributed more densely in the cranial part of the phrenic motor pool (C3), whereas motor neurons innervating the crural part are more caudal (C5)9. Furthermore, PhMN are clustered variously in the ventral horn gray matter. At C3 level, the clusters of phrenic cells lie laterally, then they shift in a ventrolateral direction and are found ventromedially at the most caudal levels10,11.
Given their vital role during inspiration, it is of the utmost importance to accurately identify PhMN in the healthy spinal cord but also follow their fate during pathological conditions, such as degenerative diseases or traumatic injuries of the spinal cord. Since PhMN do not differ morphologically from other cervical motor neurons, identification of PhMN relies on the targeted delivery of neuroanatomical tracers either at the level of primary respiratory centers8, or at the diaphragm NMJ7 or in the phrenic nerve4. The tracer is taken up by the nerve fibers and carried up to the phrenic cell bodies in the cervical spine, where it can be visualized using direct or indirect detection systems. Retrograde or anterograde tracers are commercially available with a broad range of conjugates. Noteworthy, each tracer is endowed with no, low or high abilities for trans synaptic tracing.
In the current study, we chose the beta subunit of the cholera toxin (CTB) functionalized with Alexa Fluor 555 (henceforth referred to as CTB-fluorophore) as a fluorescent label, allowing a direct visualization of PhMN on frozen spinal cord sections. CTB is usually described as a monosynaptic tracer although experimental data tend to show a transneuronal passage12. CTB has the ability to bind the ganglioside GM1 at the plasma membrane of the nerve ending. CTB is internalized via clathrin-dependent or -independent mechanisms and traffics through the trans-Golgi network into the endoplasmic reticulum in a retrograde fashion13,14. The internalization and retrograde transport seem to be dependent on the actin cytoskeleton15,16 as well as on the microtubule network17.
To demonstrate the usefulness of CTB as a retrograde neuroanatomical tracer labeling diaphragm-PhMN circuitry, CTB-fluorophore was delivered intrapleurally. CTB was administered using two techniques: the first one included a laparotomy and multiple transdiaphragmatic injections; the second one, less invasive, used a unique transthoracic injection. Four days later, fluorescently-labeled PhMNs were quantified in the cervical spinal cord from both from healthy and from spinally-injured (C4) animals.

<table>
	<tbody>
		<tr>
			<td>Glass-bead sterilizer Steri 250</td>
			<td>Keller</td>
			<td>31-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Small scissors</td>
			<td>F.S.T.</td>
			<td>14058-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Soft tweezers</td>
			<td>F.S.T.</td>
			<td>11042-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Swann Morton</td>
			<td>No.11 or 15</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera toxin subunit beta conjugated to Alexa Fluor 555</td>
			<td>Life Technologies</td>
			<td>C22843</td>
			<td>Bring at room temperature before use&#160;</td>
		</tr>
		<tr>
			<td>10ul Hamilton syringue, removable needle</td>
			<td>Sigma-Aldrich</td>
			<td>701RN</td>
			<td></td>
		</tr>
		<tr>
			<td>33-gauge needle for Hamilton syringue, 20mm length, point style 4</td>
			<td>Filter Service</td>
			<td>7803-05</td>
			<td></td>
		</tr>
		<tr>
			<td>500ul insulin syringue MyJector, 27-gauge</td>
			<td>Terumo</td>
			<td>BS05M2713</td>
			<td></td>
		</tr>
		<tr>
			<td>Orientable LED lamp</td>
			<td>V.W.R.</td>
			<td>631-0995</td>
			<td></td>
		</tr>
		<tr>
			<td>Resorbable 4/0 sutures</td>
			<td>S.M.I. AG</td>
			<td>15151519</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>F.S.T.</td>
			<td>12002-14</td>
			<td></td>
		</tr>
		<tr>
			<td>9mm autoclips</td>
			<td>Bioseb</td>
			<td>205016</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclip 9mm applier</td>
			<td>Bioseb</td>
			<td>MikRon 9mm</td>
			<td></td>
		</tr>
	</tbody>
</table>

retrograde neuroanatomical tracing of phrenic motor neurons in mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Male C57bl/6J mice (n=18), aged from 3 to 4 months were included in the study. At day 0 of the experiment, 8 mice underwent a unilateral C4 contusion, right-sided, according to published protocol7,18. As sham procedure, 10 mice underwent a laminectomy on top of C4 without contusion. At day 3, mice were prepared for the intrapleural injections of CTB-fluorophore according to the two different procedures described above. At day 7, a...

Watch this Scientific Journal Video about Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice at JoVE.com

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

The overall goal of this procedure is to identify phrenic motor neurons in mice using intrapleural delivery of fluorophore-conjugated cholera toxin subunit beta. Two techniques are shown in order to target the pleural cavity, a transdiaphragmatic and a transthoracic approach. This method can help answer key questions in the neuroscience field such as the distribution of the phrenic motor neurons, their morphology as well as the extension of the neural network.The main advantage of the procedure is that the visualization of phrenic motor neurons relies on the accumulation of the fluorescent neuroanatomical tracer that has been retrotransported from diaphragm neuromuscular junctions located in the pleural space avoiding invasive procedure in the central nervous system. Visual demonstration of this method is critical as the steps describing palpation and transthoracic injection are difficult to learn because it is difficult to describe with words the act of feeling with the hand and the dynamic of needle insertion through the thoracic wall. Sterility is essential to the surgery.Use an autoclave or glass bead sterilizer to clean the tools and prepare a clean bench coat. After anesthetizing the animal, check for the absence of reflex and apply ophthalmic ointment. Then shave the ventral skin for the transdiaphragmatic procedure.Alternatively, shave the right-sided thoracic skin for transthoracic procedure. Shave well and broad enough to prevent hair getting into the surgical field. After removing the hair, ensure aseptic conditions by scrubbing the skin with alternating application of 70%ethanol and Betadine.Be careful to scrub from the center of the site toward the periphery. Lastly, ensure that the mouse maintains its body temperature by placing it over a homeothermic pad. For each transdiaphragmatic injection, load 7.5 microliters of CTB solution in a sterile 10 microliter microsyringe with a blunt or short bevel 33 gauge needle.Then with the mouse supine, place a half an inch thick roll of gauze under its neck. Now using a scalpel blade, incise the ventral skin along the midline from the xiphoid process to the umbilical region. To avoid damage to the underlying organs, keep the skin taut and do not apply too much pressure.Next, using small scissors, carefully detach the skin from the abdominal muscles around the incision. This will make it easier to close the incision. Now perform a laparotomy.Open the abdominal cavity using small scissors to make a button-hole incision at the level of the umbilicus. Then incise the abdominal muscles along the white line up to the xiphoid process. To view the abdominal surface of the diaphragm, retract the abdominal muscles using commercial or home-made retractors.Stretch out both sides of the laparotomy and tape down the retractors to the bench coat. Then using an LED lamp, illuminate the abdominal surface of the diaphragm. Next, use soft tweezers to lift up the xiphoid appendix and pull down the lateral lobes of the liver.With small scissors, carefully cut off the suspensory ligament without damaging the gallbladder or the diaphragm. Now inject the three target sites in the right hemi-diaphragm to respectively cover the sternal, the medial and the crural regions. To avoid puncturing the lungs, only penetrate one to two millimeters beyond the diaphragm sheet.Deliver 1/3 of the volume to each of the three sites. To close the laparotomy site, suture the abdominal muscles with interrupted resorbable 4-0 sutures placed every three millimeters. Then staple the skin closed with nine millimeter wound clips placed every five millimeters.Be certain to tighten the staples to prevent the animal from pulling them off, but not so much that they impair healing. For each transthoracic injection, load a sterile 500 microliter insulin syringe with 20 microliters of CTB solution and use a beveled 27 gauge needle. Now position the mouse lateral decubitus and palpate to find the sixth and seventh ribs under the elbow region.While an assistant extends the right fore and hind limbs, insert the syringe under the sixth or the seventh rib tangentially toward the cranium and with the bevel down. Then penetrate three millimeters. Next, elevate the needle gently and the rib should lift upward if the needle is well-positioned in the thoracic cavity.Then stabilize the animal's breathing movements using two fingers to apply light pressure on the chest wall. Now deliver the full bolus of CTB solution in a single injection. Make sure to insert the needle tangentially three to four millimeters in the pleural cavity so that when you raise the needle, the ribs also lift.If you're not sure, remove the needle and do it again. After the injection, immediately position the mouse on the heating pad in a right lateral decubitus orientation. This will help the tracer to spread on the right side of the pleural cavity and it allows for monitoring of the animal's breathing.Next, administer one milliliter of sterile saline solution subcutaneously and provide followup injections if the animal appears dehydrated or listless. Also, subcutaneously administer Buprenorphine twice a day for the first two post-operative days to minimize any potential pain. Three to four-month-old male C57 black 6J mice underwent the described procedure.Three days before, half of these mice underwent a unilateral right-sided C4 spinal contusion or a laminectomy without contusion. One week after the CTB injection, whole spinal cords were harvested for histology. Motor neurons in the ventral horn were identified as a linear column of labeled cells from C3 to C5.These cells were located in the ipsilateral gray matter and looked like motor neurons.In injured animals, a striking loss of labeled neurons was observed at C4 level together with spinal tissue disruption. Next, the CTB-positive phrenic motor neurons were manually counted in every fifth transverse section. The total number of labeled phrenic motor neurons per hemi-cord was slightly lower when using the transdiaphragmatic approach than the transthoracic approach, but this difference was not significant.However, the number of labeled neurons were halved following the C4 injury and this was significant. While attempting this procedure, it is important to remember to consider the animal welfare and to take measures to minimize the pain and discomfort. Following this procedure, other methods like anterograde neuroanatomical tracing or transsynaptic tracing studies can be performed in order to answer additional questions regarding the connectivity of phrenic motor neurons and afferent projection fibers from the rostral ventral respiratory group.Though this method can provide insight into morphological, biochemical, developmental, and functional aspects of phrenic motor neurons, it can also be used to study phrenic circuits in mouse models of amyotrophic lateral sclerosis, spinal muscular atrophy, and spinal cord injury.

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